Whole genome amplification — applications and advances

doi:10.1016/S0958-1669(02)00286-0

Current Opinion in Biotechnology

Volume 13, Issue 1, 1 February 2002, Pages 65-67

https://doi.org/10.1016/S0958-1669(02)00286-0 Get rights and content

Abstract

The concept of whole genome amplification is something that has arisen in the past few years as the polymerase chain reaction (PCR) has been adapted to replicate regions of genomes that are of biological interest. The applications are many — forensic science, embryonic disease diagnosis, bioterrorism genome detection, ‘immortalization’ of clinical samples, microbial diversity, and genotyping. Several recent papers suggest that whole genomes can be replicated without bias or non-random distribution of the target, these findings open up a new avenue to molecular biology.

Introduction

The basic premise of molecular biology is that you are working on DNA. A simple fact, but in many cases perhaps the hardest starting point to achieve. The phrase ‘I lost my DNA’ was about as common as ‘Anyone want to get a beer?’ when I was a graduate student! Today, kits for DNA manipulation and clean up are both abundant and ‘almost’ fail safe, but the issue of obtaining good, clean DNA to start with is still the most difficult step.

Over the past ten years, methods for DNA amplification have been described such as degenerate oligonucleotide primed PCR (DOP–PCR) 1., 2. and primer extension preamplification (PEP) [3], which have been used as precursors to a variety of genetic tests and assays. Neither DOP–PCR nor PEP claim to replicate the target DNA in its entirety [2] or provide a complete coverage of particular loci [4]. Instead, these protocols produce an amplified source for genotyping or marker identification of some type. It is also the case that the product produced via these methods is short (<3 kb) and therefore cannot be used in many applications [1]. These approaches have proven to be vital in the fields of forensics and genetic disease diagnosis where DNA quantities are limited, but many tests are required to investigate few markers or loci. In this review we discuss recent advances in this technology and potential applications of whole genome amplification.

Section snippets

Applications of whole genome amplification

One recent example of the widespread use of these approaches is in preimplantation genetics and diagnosis (PGD). This is the basic technique used to infer the status of an embryo from a single cell biopsied 2–3 days after fertilization. Several tests have been described that assay single loci for defects including one that uses multiplex fluorescent PCR to detect trinucleotide amplification in the myotonic dystrophy locus [5]. In a recent paper by Wells and Delhanty [6•], whole genome

Strand-displacement amplification

A new method for whole genome amplification has been introduced in the past year and holds a great deal of promise. The method is based upon the strand-displacement amplification approach used in rolling circle amplification [8•]. Kits are now available for the amplification of plasmid templates for DNA sequencing (TempliPhi, Amersham Biosciences) and whole genome amplification systems for research and diagnostic uses are due to follow soon (J Nelson, S Kingsmore, personal communication). The

Microbial genome amplification

In the field of microbial diversity, the vast majority (perhaps greater than 80%) of microbes cannot be cultured and many live in communities of multiple organisms. The use of strand-displacement amplification could be used to amplify DNA from soil or water samples or from flow-sorted samples in the case of microbial communities. The DNA obtained could then be used for DNA sequencing or rapid identification methods. Detter et al. [13••] used this technique to amplify DNA directly from

Conclusions

The applications of whole genome amplification, whether using DOP, PEP or strand-displacement amplification, are gaining ground and opening up new scientific approaches. The ability to take individual cells and generate sufficient, renewable DNA to perform a multitude of genetic tests and assays is the dream of many molecular biologists. For the identification of markers or gross changes in DNA structure, DOP and PEP offer significant advantages and are already in use by large-scale academic

Acknowledgements

This work was performed under the auspices of the US Department of Energy, Office of Biological and Environmental Research, and the University of California, under Contracts No. W-7405-Eng-48, No. DE-AC03-76SFOO098, and No. W-7405-ENG-36.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest
•• of outstanding interest

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Effectiveness of whole genome amplification prior to short tandem repeat analysis for degraded DNA
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Short tandem repeat (STR) analysis is prone to failure as DNA is frequently damaged by various environmental factors; hence, increasing the number of starting templates may constitute a feasible approach to improve STR profiling success. Whole genome amplification (WGA) is often applied to bolster starting template quantity. Moreover, WGA can reportedly be used on degraded DNA samples in forensics. Therefore, we utilized a PCR-based WGA method, termed “modified improved primer extension preamplification” (mIPEP), prior to STR analysis of degraded DNA, as this method is less affected by DNA quantity and quality than most others. Saliva from four volunteers was dried on glass fiber filter papers (paper) and glass slides (glass) and irradiated with UVA light (365 nm). The mIPEP method was initiated using 5, 0.5, and 0.05 ng of DNA following DNA extraction. The DNA degradation index (DI) was calculated based on the ratio of 129 to 41 bp DNA fragments; lower numbers indicate higher degradation. Following mIPEP, STR analysis was performed using the AmpFlSTR Identifiler PCR amplification kit. The number of detectable STR loci, with and without mIPEP, decreased according to reduced DI in a different manner for the various DNA concentrations extracted from paper and glass. Specifically, for the 5 ng DNA sample on paper, at a DI < 0.2, the number of detectable STR loci was greater with mIPEP than without it, owing to fewer locus drop-outs. Similarly, the 0.05 ng DNA sample deposited on paper, at DI ≥ 0.7, exhibited higher numbers of detectable STR loci when prepared using mIPEP owing to fewer allele drop-outs. Moreover, among samples deposited on glass, the 0.05 ng DNA sample at DI ≥ 0.4 afforded a larger number of detectable STR loci when prepared using mIPEP than those without mIPEP, owing to fewer locus drop-outs. These findings suggest that performing mIPEP in accordance with sample DNA condition (e.g., quantity and quality) may lead to increased success of STR analysis. Notably, the conditions identified as most responsive to mIPEP were consistent across both UVA-irradiated and environmentally-damaged sample states. Taken together, our results suggest that applying mIPEP would be beneficial to obtain improved STR profiles under conditions involving severely degraded samples with large quantities of DNA, or with small quantities of DNA albeit with slight degradation.
Improved molecular karyotyping in glioblastoma
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Citation Excerpt :
MDA is excellent for detecting single nucleotide variants (SNVs) because Ф29 exhibits proofreading activity, but also amplifies promiscuous annealing events that create fusion artifacts and uneven DNA synthesis [38]. Such artifacts increase the number of unmapped reads and the variance in the median absolute deviation (MAD) of normalized counts between neighboring bins, which is used to estimate integer copy states [17,20,30,39,40]. DOP-PCR detects structural features better than MDA, but SNV detection is diminished because of higher error rates [20].
Uneven replication creates artifacts during whole genome amplification (WGA) that confound molecular karyotype assignment in single cells. Here, we present an improved WGA recipe that increased coverage and detection of copy number variants (CNVs) in single cells. We examined serial resections of glioblastoma (GBM) tumor from the same patient and found low-abundance clones containing CNVs in clinically relevant loci that were not observable using bulk DNA sequencing. We discovered extensive genomic variability in this class of tumor and provide a practical approach for investigating somatic mosaicism.
Colorimetric biosensor based on a DNAzyme primer and its application in logic gate operations for DNA screening
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Citation Excerpt :
Various types of PCR technology, such as the multiple PCR, nested PCR, isothermal amplification, as well as the introduction of quantitative PCR (qPCR) and digital PCR (dPCR), have therefore been applied to detect, screen, and quantify targets [1–6]. However, PCR results have to be analyzed based on amplicon length, or the monitoring of fluorescent change, thus complicating analysis for point-of-care diagnosis [7–12]. Recent biotechnological developments, such as optical, electronic, and microgravimetric platforms, to offer a variety of alternative choices for interpreting result [13–15].
A colorimetric biosensor for DNA screening was designed based on the conformational changes of the horseradish peroxidase (HRP)-mimicking DNAzyme. The scheme of DNA biosensing was designed based on the base pairing of DNAzyme sequence to inhibit the formation of HRP-mimicking hemin/G-quadruplex structures in the process of amplification. DNA could be amplified via the universal primer multiplex polymerase chain reaction (UP-M-PCR) and innovatively detected as color disappear in the reaction visible to the naked eye. The input of key factors and the output of optical characteristics in the reaction inspired the development of an OR logic gate operation for DNA detection. This biosensor overcomes self-inhibition and amplification disparity with the help of UP-M-PCR, thereby exhibiting high specificity and high-throughput without the requirement of gel analysis work. This biosensing system also presented 1% sensitivity and approximately 180 copy numbers in triplicate. The biosensor was used to screen elements from genetically modified organisms (GMOs) and covered more than 90% of all globally authorized events in the world. The designed colorimetric biosensor is a rapid, portable and versatile tool for nucleic acids detection and diagnosis in the field.
Development and validation of a whole genome amplification long-range PCR sequencing method for ADPKD genotyping of low-level DNA samples
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Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in two large genes, PKD1 and PKD2, but genetic testing is complicated by the large transcript sizes and the duplication of PKD1 exons 1–33 as six pseudogenes on chromosome 16. Long-range PCR (LR-PCR) represents the gold standard approach for PKD1 genetic analysis. However, a major issue with this approach is that it requires large quantities of genomic DNA (gDNA) material limiting its application primarily to DNA extracted from blood. In this study, we have developed a whole genome amplification (WGA)-based genotyping assay for PKD1 and PKD2, and examined whether this approach can be applied to biosamples with low DNA yield, including blood, buccal cells and urine. DNA samples were amplified by multiple displacement amplification (MDA) and a high-fidelity DNA polymerase followed by LR-PCR and exon-specific amplifications of PKD1 and PKD2 respectively, and Sanger sequencing. This method has generated large amounts of DNA with high average product length (> 10 kb), which were uniformly amplified across all sequences assessed. When compared to the gDNA direct sequencing method for six ADPKD samples, a total of 89 variants were detected including all 86 variations previously reported, in addition to three new variations, including one pathogenic mutation not previously detected by the standard gDNA-based analysis. We have further applied WGA to ADPKD mutation analysis of low DNA-yield specimens, successfully detecting all 63 gene variations. Compared to the gDNA method the WGA-based assay had a sensitivity and specificity of 100%. In conclusion, WGA-based LR-PCR represents a major technical improvement for PKD genotyping from trace amounts of DNA.
Application of next-generation sequencing technology in forensic science
2014, Genomics, Proteomics and Bioinformatics
Citation Excerpt :
Using NGS technology, millions of miRNA sequences can be rapidly analyzed to identify organ- and developmental stage-specific expression, as well as miRNA expression in different disease states, thus providing a powerful tool for forensic analysis. In practical forensic science, DNA samples are usually limited and often cannot fulfill the requirements of simultaneously analyzing multiple loci on different chromosomes in mitochondrial genome [67–69]. This may result in difficulties in providing sufficient information and can limit their use as legal evidence.
Next-generation sequencing (NGS) technology, with its high-throughput capacity and low cost, has developed rapidly in recent years and become an important analytical tool for many genomics researchers. New opportunities in the research domain of the forensic studies emerge by harnessing the power of NGS technology, which can be applied to simultaneously analyzing multiple loci of forensic interest in different genetic contexts, such as autosomes, mitochondrial and sex chromosomes. Furthermore, NGS technology can also have potential applications in many other aspects of research. These include DNA database construction, ancestry and phenotypic inference, monozygotic twin studies, body fluid and species identification, and forensic animal, plant and microbiological analyses. Here we review the application of NGS technology in the field of forensic science with the aim of providing a reference for future forensics studies and practice.
A novel whole genome amplification method using type IIS restriction enzymes to create overhangs with random sequences
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Current Opinion in Biotechnology

Abstract

Introduction

Section snippets

Applications of whole genome amplification

Strand-displacement amplification

Microbial genome amplification

Conclusions

Acknowledgements

References and recommended reading

References (13)

Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer

Genomics

Multiple mutation analyses in single tumor cells with improved whole genome amplification

Am J Pathol

Fidelity of φ29 DNA polymerase. Comparison between protein-primed initiation and DNA polymerization

J Biol Chem

High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus

Gene

Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genotypes to be performed on less than one nanogram of genomic DNA

Proc Natl Acad Sci USA

Whole genome amplification from a single cell: implications for genetic analysis

Proc Natl Acad Sci USA

Cited by (69)

Effectiveness of whole genome amplification prior to short tandem repeat analysis for degraded DNA

Improved molecular karyotyping in glioblastoma

Colorimetric biosensor based on a DNAzyme primer and its application in logic gate operations for DNA screening

Development and validation of a whole genome amplification long-range PCR sequencing method for ADPKD genotyping of low-level DNA samples

Application of next-generation sequencing technology in forensic science

A novel whole genome amplification method using type IIS restriction enzymes to create overhangs with random sequences

ReviewWhole genome amplification — applications and advances

Abstract

Introduction

Section snippets

Applications of whole genome amplification

Strand-displacement amplification

Microbial genome amplification

Conclusions

Acknowledgements

References and recommended reading

Genomics

Am J Pathol

J Biol Chem

Gene

Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genotypes to be performed on less than one nanogram of genomic DNA

Proc Natl Acad Sci USA

Whole genome amplification from a single cell: implications for genetic analysis

Proc Natl Acad Sci USA

Review
Whole genome amplification — applications and advances